title: HER2-specific affibody-conjugated thermosensitive liposomes (Affisomes) for improved delivery of anticancer agents.
abstract: Thermosensitive liposomes are attractive vehicles for the delivery and release of drugs to tumors. To improvethe targeting efficacy for breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (Z(HER2:342)-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as "Affisomes" were investigated. Thermosensitive small unilamellar vesicle (SUV) liposomes of (80-100 nm) a diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41 degrees C) as the matrix lipid and a maleimide-conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (e.g., tributylphosphine) for 16-20 hours under an argon atmosphere. Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4-12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were approximately 70% at a protein-lipid ratio of 20 microg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped calcein (a water-soluble fluorescent probe, lambdaex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90-100%) at 41 degrees C. In contrast, liposomes prepared from Egg phosphatidyl choline (Egg PC, Tm approximately 0 degrees C) under similar conditions released only 5-10% calcein at 41 degrees C. Affisomes, when stored at room temperature, retained > 90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in phosphate-buffered saline, supplemented with 10% heat-inactivated serum (fetal bovine serum) did not result in a destabilization of liposomes. Therefore, Affisomes present promising, novel drug-delivery candidates for breast cancer targeting.
authors.affiliation: CCR Nanobiology Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702-1201, USA. apuri@helix.nih.gov
authors.forename: Anu
authors.initials: A
authors.lastname: Puri
authors.forename: Gabriela
authors.initials: G
authors.lastname: Kramer-Marek
authors.forename: Ryan
authors.initials: R
authors.lastname: Campbell-Massa
authors.forename: Amichai
authors.initials: A
authors.lastname: Yavlovich
authors.forename: Shrikant C
authors.initials: SC
authors.lastname: Tele
authors.forename: Sang-Bong
authors.initials: SB
authors.lastname: Lee
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Clogston
authors.forename: Anil K
authors.initials: AK
authors.lastname: Patri
authors.forename: Robert
authors.initials: R
authors.lastname: Blumenthal
authors.forename: Jacek
authors.initials: J
authors.lastname: Capala
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage*; D002417:Cattle; D004337:Drug Carriers / Q000737:chemistry*; D016503:Drug Delivery Systems; D008081:Liposomes / Q000737:chemistry*; D015394:Molecular Structure; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013696:Temperature
chemical_list: D000970:Antineoplastic Agents; D004337:Drug Carriers; D008081:Liposomes; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C533351:affibody (ZHER2-342)2